The trehalose phosphate synthetase of Mycobacterium smegmatis is an unusual glycosyl transferase in that it can utilize all of the common glucose sugar nucleotides as substrates. Perhaps more interesting is the fact that the enzyme requires a high molecular weight polyanion such as heparin chondroitin sulfate or RNA as an activator when pyrimidine nucleotides (UDPG, TDPG, CDPG) are used as substrates, but does not require this polyanion in order to use purine nucleotides (ADPG, GDPG). We have now purified this enzyme to homogeneity using a combination of DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and affinity chromatography on a column of heparin-Sepharose. The purified enzyme also utilizes UDPG and GDPG indicating that these activities reside in the same protein. We are using this purified enzyme to study subunit composition and aggregation and plan to do binding studies to determine optimum size and charge of polyanion for binding. We also plan to determine whether this optimum size for binding correlates with activation of the enzyme. BIBLIOGRAPHIC REFERENCES: Elbein, A.D. and Mitchell, M. Protein Polyanion Interactions. Arch. Biochem. Biophys., 168, 369 (1975). Chambers, J. and Elbein, A.D. Biosynthesis and Characterization of Lipid-linked Sugars and Glycoproteins in Aorta. J. Biol. Chem. 250, 6904 (1975).